Atom probe tomography (APT) is an unusual and emerging imaging and microanalysis technique enabling three-dimensional atomic-scale mapping of chemical composition in materials. Thus far, APT has been applied almost exclusively to rigid inorganic materials. Samples are fabricated into needle-like specimens terminated with extremely sharp tips. Specimens are formed either from bulk materials, or in some instances machined from more complex objects such as semiconductor devices.
These specimens are then cryogenically cooled and analyzed in vacuum using highly controlled atom-by-atom field evaporation from the tip surface. Atoms leave the quasi-spherical tip as ions, are accelerated by a high electric field away from the tip, and impinge on an ion detector. This detector has single-ion sensitivity, along with high spatial and temporal resolution. Radial projection of ions from tip to the detector plane is used to obtain the initial position of atoms or molecules on the tip surface. Simultaneously, time-of-flight measurements permit very precise determination of the mass-to-charge (m/z) ratio of individual ions. The resultant data sets of these ion detections permit three-dimensional reconstruction of the atomic structure of the original needle-like specimen volume.
It is easy to see that this is a completely destructive process. Field evaporation of individual surface ions is achieved at a precise time by either electrical pulsing, or with the assistance of short-duration laser pulses focused onto the tip. The latter method permits the analysis of samples with low electrical conductivity. Within the constraints of this unusual specimen geometry and complete disintegration of the specimen into individual ions, APT can be regarded as the ultimate microanalysis technique.
APT can be traced back to the pioneering Field Ion Microscopy (FIM) experiments of E. W. Müller in the 1950s. At his Pennsylvania State University laboratory, individual atoms forming the lattice of refractory-metal tips were directly imaged for the first time. In 1967, atom probe field ion microscope (APFIM) was also developed in this laboratory as an extension of the capabilities of the basic field ion microscopy (FIM) instrument. Using APFIM, single surface atoms individually preselected in real-time from a FIM image of a specimen tip could be chemically identified.
This is accomplished by pulsed field evaporation of the selected atom (along with other atoms) and having the preselected atom pass through a hole in the phosphor imaging screen. APFIM analysis is accomplished by determining the mass-to-charge (m/z) ratio of the preselected atom along with a time-of-flight mass spectrometer having single-ion sensitivity. However, the analyzing speed of this early APFIM technology was extremely slow, and only a miniscule fraction of the atoms comprising the specimen could be identified. The vast majority of field evaporated atoms were simply deposited without detection on the FIM phosphor screen.
Later in the 1970s with the introduction of more advanced microchannel plate (MCP) detectors, the 10-cm Atom Probe and Imaging Atom Probe (IAP) were developed by J. A. Panitz. This permitted many atoms to be analyzed simultaneously from a tip. A combination of APFIM and IAP eventually evolved into current APT methodology starting in the 1980s. Modern commercial instruments, using a modified configuration known as local-electrode atom probe (LEAP®) are capable of very high detection efficiency of specimen atoms, and can analyze close to 107 cubic nanometers (nm3) of tip material per day.
A schematic diagram of the operating principles of a prior art APT instrument 10 using a local-electrode configuration is shown in FIG. 1. FIG. 1 shows a specimen 12 cooled to cryogenic temperatures, typically 20-50° K at a high positive potential 14, typically 2-15 kilovolts (kV) from the ground. Tip 30 of specimen 12 is extremely sharp and created either through electrochemical etching or using a focused ion beam (FIB) instrument. It typically has a tip-radius of 20-100 nm. FIG. 1 of the prior art which is not drawn to scale for explanatory purposes also shows a local-electrode (or local electrode) 16 held at or near-ground potential 18 with respect to specimen 12.
Local-electrode 16 has a funnel shape, with a small opening aperture of several tens of microns. This aperture is positioned a comparable distance from specimen tip 30. The use of local-electrode 16 greatly reduces the voltage required for field evaporation due to enhanced field strength as a result of the small gap/distance between electrode 16 and tip 30.
Specifically, electrode 16 is pulsed slightly negatively with respect to its direct current (DC) near-ground potential 18 in sub-ns duration pulses so as to precisely control the release of ions from tip 30 by field evaporation. In FIG. 1, reference numeral 19 depicts a single square-wave pulse used for the above-mentioned slightly negative pulses for electrode 16.
Instead of using slightly negative pulses with respect to ground potential 18, pulsed laser focused on tip 30 may also be used to release ions under field evaporation. Such a laser pulse 24 is shown in FIG. 1. In such a scenario, local-electrode 16 is connected to a steady ground potential 18 to provide nearly the electric field threshold necessary for guiding the release of ions by field evaporation. Thus, under the influence of either a pulsed negative potential of local-electrode 16 or laser pulses 24, atoms on tip 30 of specimen 12 are ionized and released by field evaporation using these two alternate methods of pulsed field evaporation.
Two such ionized atoms 26 and 28 are shown in FIG. 1. The former is a copper Cu+ ion and the latter an iron or Fe++ ion, shown here being released from a representative sample alloy of iron and copper. Mode-locked solid-state lasers are typically employed for the above application, which can have pulse durations of under 1 picosecond (ps), and pulse rates in the hundreds of kilohertz (kHz). This can permit very precise timing of over a thousand field evaporated ions/second. Typically, operating parameters are adjusted so that single ions are created by only about 1% of pulses to avoid multiple ions being created in each individual pulse.
Ions 26 and 28 liberated from tip 30 of specimen 12 travel through local-electrode 16 near ground potential 18 and impinge on a particle detector. The particle detector shown in FIG. 1 comprises a microchannel plate (MCP) 20 that has a high enough gain to permit single particle sensitivity along with high spatial resolution due to its multiplicity of channels. The particle detector shown also includes a cross-delay line position sensitive detector 22. Ions impinging on microplate 20 produce many secondary electron emissions which are detected by cross-delay line position detector 22 for determining the spatial position of the MCP electron output emissions. Together, microplate 20 and detector 22 are able to provide the time-of-flight (TOF) measurements of incoming ions 26 and 28 as well as their spatial resolution (Xs, Ys) on detector 22. In some atom probe instruments, an ion optical system known as a “reflectron” is inserted into the flight path of the ions to increase transit time for achieving better mass resolution.
After collecting typically many millions of such individual emissions, sophisticated reconstruction software is used to reconstruct a three-dimensional (3-D) atomic-scale map of tip 30 of original sample 12 using the large data sets of field evaporated ions. The resultant full three-dimensional atomic-scale map of the tip can be manipulated for optimal viewing of material structure and composition at sub-nm spatial resolution. FIG. 2 shows a two-dimensional cross-sectional image 40 from such a 3-D atomic scale map of a reconstructed tip, such as tip 30 of specimen 12 of FIG. 1. More specifically, FIG. 2 shows a reconstructed tip that has undergone APT from a sample of a steel alloy, with manganese (Mn), nickel (Ni), and titanium (Ti) impurities, and published in NPL reference “Chemical gradients across phase boundaries between martensite and austenite in steel studied by atom probe tomography and simulation”, by Dmitrieva et. al of Max-Planck Institute, Dusseldorf, Germany, dated October 2010.
Let us now discuss the mounting member of such a prior art APT instrument that is responsible for holding specimen 12 of FIG. 1. For a local-electrode APT configuration, such a mounting member is typically known as the “puck”. Such a puck 60 of the prior art is shown in FIG. 3. More specifically, puck assembly 60 consists of a casing 62 and mount 64 typically made out of copper. At one end of the casing is a locking pin mechanism consisting of a mounting pin 68 and lock 70. A transfer arm/shuttle with a load-lock mechanism (not shown) is used to move the puck into the cryogenic and UHV analysis chamber and lock it in place with the help of mounting pin 68 and lock 70.
Casing 62 is also typically made out of copper to facilitate good thermal and electrical conduction with mount 64 containing specimen 12.
Electrical connection to specimen 12 as explained earlier in reference to FIG. 1 is provided by conduction through copper casing 62 and mount 64. Specimen 12 is locked into mount 64 by employing a set screw 66 shown. In other variations of APT the mounting member for the specimen may be different than this puck. Potential options include a structure resembling a “stub” used for mounting a sample in a scanning electron microscope (SEM). As will be explained in the summary and detailed description sections and the accompanying figures, the present design provides key innovations over specimen holding designs in general and puck 60 in particular of the prior art.
Previous APT Work with Biological Specimens
It has long been a dream of scientific community to extend field ion microscopy (FIM), and later atom probe tomography (APT) to biological samples for attaining atomic scale/resolution imaging of biological materials. Unfortunately, attempts to realize this have met with limited success due to a number of formidable technical challenges. Thus far, hard mineralized biological materials have been best suited for the technique. Difficulties with softer materials include both specimen preparation issues, as well as physical complications intrinsic to FIM and APT analysis of delicate and complex bio-samples.
The above complications include asymmetrical field evaporation from highly heterogeneous biological materials and extremely high and potentially destructive stress of the electrostatic field on the relatively fragile specimen tips during imaging and analysis. These complications further include the need to maintain the biological materials in a high vacuum as well as cryogenic environment. As a result, the goal of routine biological APT of biological specimens or bio-samples remains elusive.
Scientific instruments for determining the structure and composition of materials at the nanoscale are crucial in modern science and technology. Sales of these extremely sophisticated instruments are a multi-billion dollar industry. They are used for both basic research applications, as well as applied development of a vast array of commercial products. As compared to electron microscopy (EM), FIB, and x-ray analysis, APT is currently a much more specialized technique, with restricted application. This is due to the need to prepare samples into very fine specimen tips, and the limited types of materials that are usable with APT. The equipment is also extremely costly, which has limited the number of installed instruments.
The potential of APT to produce useful imaging of ultrastructure of biological materials could be a tremendous advancement to this form of imaging and chemical analysis. As with inorganic samples, APT holds the promise of atomic-scale images of biological samples, along with chemical identification. Such capability is not achievable with any existing technique known in the prior art. The present invention transforms biological APT to become an extremely valuable complementary tool, when used in conjunction with other techniques, such as transmission electron microscopy (TEM). In addition to biological materials, other soft materials such as polymers would also find useful application of the present technology.
Thus, the present invention is primarily but not exclusively directed to methods and apparatus for applying APT to biological materials and addresses shortcomings of the prior art in handling and analyzing soft materials. As will be taught, the instant inventive approach includes producing tip structures that differ from those fabricated using focused ion beam (FIB) techniques of the prior art. The instant tip structures facilitate stable field evaporation in a manner akin to that observed with inorganic materials and thus provides accurate reconstruction of large specimen volumes without tip failure. The instant techniques are also less time-consuming and less costly to implement than the fabrication techniques of the prior art. Both polymer-embedded and frozen-hydrated material may be applicable for use with the instant techniques.
The benefits of the capabilities of APT for biological applications would also have positive ramifications for many areas of life science. Both basic research in the life sciences, as well as medical applications stands to benefit. This will greatly increase the use of APT, and increase the commercial potential of these sophisticated instruments. With higher demand, the cost of these instruments could also significantly decrease. At present, the restricted types of materials applicable to the technique and high cost of the instruments have limited the total commercial market to approximately 100 atom-probe instruments of all kinds worldwide. Expanding APT into the biological realm could greatly increase the use and commercial sales of these instruments.